Biologically Enhanced Genome-Wide Association Study Provides Further Evidence for Candidate Loci and Discovers Novel Loci That Influence Risk of Anterior Cruciate Ligament Rupture in a Dog Model.

Anterior cruciate ligament (ACL) rupture is a common condition that disproportionately affects young people, 50% of whom will develop knee osteoarthritis (OA) within 10 years of rupture. ACL rupture exhibits both hereditary and environmental risk factors, but the genetic basis of the disease remains unexplained. Spontaneous ACL rupture in the dog has a similar disease presentation and progression, making it a valuable genomic model for ACL rupture. We leveraged the dog model with Bayesian mixture model (BMM) analysis (BayesRC) to identify novel and relevant genetic variants associated with ACL rupture. We performed RNA sequencing of ACL and synovial tissue and assigned single nucleotide polymorphisms (SNPs) within differentially expressed genes to biological prior classes. SNPs with the largest effects were on chromosomes 3, 5, 7, 9, and 24. Selection signature analysis identified several regions under selection in ACL rupture cases compared to controls. These selection signatures overlapped with genome-wide associations with ACL rupture as well as morphological traits. Notable findings include differentially expressed ACSF3 with MC1R (coat color) and an association on chromosome 7 that overlaps the boundaries of SMAD2 (weight and body size). Smaller effect associations were within or near genes associated with regulation of the actin cytoskeleton and the extracellular matrix, including several collagen genes. The results of the current analysis are consistent with previous work published by our laboratory and others, and also highlight new genes in biological pathways that have not previously been associated with ACL rupture. The genetic associations identified in this study mirror those found in human beings, which lays the groundwork for development of disease-modifying therapies for both species.

Mapping of the contraction-induced phosphoproteome identifies TRIM28 as a significant regulator of skeletal muscle size and function.

Mechanical signals, such as those evoked by maximal-intensity contractions (MICs), can induce an increase in muscle mass. Rapamycin-sensitive signaling events are widely implicated in the regulation of this process; however, recent studies indicate that rapamycin-insensitive signaling events are also involved. Thus, to identify these events, we generate a map of the MIC-regulated and rapamycin-sensitive phosphoproteome. In total, we quantify more than 10,000 unique phosphorylation sites and find that more than 2,000 of these sites are significantly affected by MICs, but remarkably, only 38 of the MIC-regulated events are mediated through a rapamycin-sensitive mechanism. Further interrogation of the rapamycin-insensitive phosphorylation events identifies the S473 residue on Tripartite Motif-Containing 28 (TRIM28) as one of the most robust MIC-regulated phosphorylation sites, and extensive follow-up studies suggest that TRIM28 significantly contributes to the homeostatic regulation of muscle size and function as well as the hypertrophy that occurs in response to increased mechanical loading.

The role of raptor in the mechanical load-induced regulation of mTOR signaling, protein synthesis, and skeletal muscle hypertrophy.

It is well known that an increase in mechanical loading can induce skeletal muscle hypertrophy, and a long standing model in the field indicates that mechanical loads induce hypertrophy via a mechanism that requires signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Specifically, it has been widely proposed that mechanical loads activate signaling through mTORC1 and that this, in turn, promotes an increase in the rate of protein synthesis and the subsequent hypertrophic response. However, this model is based on a number of important assumptions that have not been rigorously tested. In this study, we created skeletal muscle specific and inducible raptor knockout mice to eliminate signaling by mTORC1, and with these mice we were able to directly demonstrate that mechanical stimuli can activate signaling by mTORC1, and that mTORC1 is necessary for mechanical load-induced hypertrophy. Surprisingly, however, we also obtained multiple lines of evidence that indicate that mTORC1 is not required for a mechanical load-induced increase in the rate of protein synthesis. This observation highlights an important shortcoming in our understanding of how mechanical loads induce hypertrophy and illustrates that additional mTORC1-independent mechanisms play a critical role in this process.-You, J.-S., McNally, R. M., Jacobs, B. L., Privett, R. E., Gundermann, D. M., Lin, K.-H., Steinert, N. D., Goodman, C. A., Hornberger, T. A. The role of raptor in the mechanical load-induced regulation of mTOR signaling, protein synthesis, and skeletal muscle hypertrophy.

A map of the phosphoproteomic alterations that occur after a bout of maximal-intensity contractions.

KEY POINTS: Mechanical signals play a critical role in the regulation of muscle mass, but the molecules that sense mechanical signals and convert this stimulus into the biochemical events that regulate muscle mass remain ill-defined. Here we report a mass spectrometry-based workflow to study the changes in protein phosphorylation that occur in mouse skeletal muscle 1 h after a bout of electrically evoked maximal-intensity contractions (MICs). Our dataset provides the first comprehensive map of the MIC-regulated phosphoproteome. Using unbiased bioinformatics approaches, we demonstrate that our dataset leads to the identification of many well-known MIC-regulated signalling pathways, as well as to a plethora of novel MIC-regulated events. We expect that our dataset will serve as a fundamentally important resource for muscle biologists, and help to lay the foundation for entirely new hypotheses in the field. ABSTRACT: The maintenance of skeletal muscle mass is essential for health and quality of life. It is well recognized that maximal-intensity contractions, such as those which occur during resistance exercise, promote an increase in muscle mass. Yet, the molecules that sense the mechanical information and convert it into the signalling events (e.g. phosphorylation) that drive the increase in muscle mass remain undefined. Here we describe a phosphoproteomics workflow to examine the effects of electrically evoked maximal-intensity contractions (MICs) on protein phosphorylation in mouse skeletal muscle. While a preliminary phosphoproteomics experiment successfully identified a number of MIC-regulated phosphorylation events, a large proportion of these identifications were present on highly abundant myofibrillar proteins. We subsequently incorporated a centrifugation-based fractionation step to deplete the highly abundant myofibrillar proteins and performed a second phosphoproteomics experiment. In total, we identified 5983 unique phosphorylation sites of which 663 were found to be regulated by MIC. GO term enrichment, phosphorylation motif analyses, and kinase-substrate predictions indicated that the MIC-regulated phosphorylation sites were chiefly modified by mTOR, as well as multiple isoforms of the MAPKs and CAMKs. Moreover, a high proportion of the regulated phosphorylation sites were found on proteins that are associated with the Z-disc, with over 74% of the Z-disc proteins experiencing robust changes in phosphorylation. Finally, our analyses revealed that the phosphorylation state of two Z-disc kinases (striated muscle-specific serine/threonine protein kinase and obscurin) was dramatically altered by MIC, and we propose ways these kinases could play a fundamental role in skeletal muscle mechanotransduction.

Identification of mechanically regulated phosphorylation sites on tuberin (TSC2) that control mechanistic target of rapamycin (mTOR) signaling.

Mechanistic target of rapamycin (mTOR) signaling is necessary to generate a mechanically induced increase in skeletal muscle mass, but the mechanism(s) through which mechanical stimuli regulate mTOR signaling remain poorly defined. Recent studies have suggested that Ras homologue enriched in brain (Rheb), a direct activator of mTOR, and its inhibitor, the GTPase-activating protein tuberin (TSC2), may play a role in this pathway. To address this possibility, we generated inducible and skeletal muscle-specific knock-out mice for Rheb (iRhebKO) and TSC2 (iTSC2KO) and mechanically stimulated muscles from these mice with eccentric contractions (EC). As expected, the knock-out of TSC2 led to an elevation in the basal level of mTOR signaling. Moreover, we found that the magnitude of the EC-induced activation of mTOR signaling was significantly blunted in muscles from both inducible and skeletal muscle-specific knock-out mice for Rheb and iTSC2KO mice. Using mass spectrometry, we identified six sites on TSC2 whose phosphorylation was significantly altered by the EC treatment. Employing a transient transfection-based approach to rescue TSC2 function in muscles of the iTSC2KO mice, we demonstrated that these phosphorylation sites are required for the role that TSC2 plays in the EC-induced activation of mTOR signaling. Importantly, however, these phosphorylation sites were not required for an insulin-induced activation of mTOR signaling. As such, our results not only establish a critical role for Rheb and TSC2 in the mechanical activation of mTOR signaling, but they also expose the existence of a previously unknown branch of signaling events that can regulate the TSC2/mTOR pathway.

Effects of oral phosphatidic acid feeding with or without whey protein on muscle protein synthesis and anabolic signaling in rodent skeletal muscle.

BACKGROUND: Phosphatidic acid (PA) is a diacyl-glycerophospholipid that acts as a signaling molecule in numerous cellular processes. Recently, PA has been proposed to stimulate skeletal muscle protein accretion, but mechanistic studies are lacking. Furthermore, it is unknown whether co-ingesting PA with other leucine-containing ingredients can enhance intramuscular anabolic signaling mechanisms. Thus, the purpose of this study was to examine if oral PA feeding acutely increases anabolic signaling markers and muscle protein synthesis (MPS) in gastrocnemius with and without whey protein concentrate (WPC). METHODS: Overnight fasted male Wistar rats (~250 g) were randomly assigned to four groups: control (CON, n = 6-13), PA (29 mg; n = 8), WPC (197 mg; n = 8), or PA + WPC (n = 8). Three hours post-feeding, gastrocnemius muscle was removed for markers of Akt-mTOR signaling, gene expression patterns related to skeletal muscle mass regulation and metabolism, and MPS analysis via the SUnSET method. RESULTS: Compared to CON rats, PA, WPC and PA + WPC resulted in a significant elevation in the phosphorylation of mTOR (Ser2481) and rps6 (Ser235/236) (p < 0.05) in the gastrocnemius though there were no differences between the supplemented groups. MPS levels in the gastrocnemius were significantly (p < 0.05) elevated in WPC versus CON rats, and tended to be elevated in PA versus CON rats (p = 0.08), though MPS was less in PA + WPC versus WPC rats (p < 0.05) in spite of robust increases in mTOR pathway activity markers in the former group. C2C12 myoblast data agreed with the in vivo data herein showing that PA increased MPS levels 51% (p < 0.001) phosphorylated p70s6k (Thr389) levels 67% (p < 0.001). CONCLUSIONS: Our results are the first in vivo evidence to demonstrate that PA tends to increases MPS 3 h post-feeding, though PA may delay WPC-mediated MPS kinetics within a 3 h post-feeding window.

Yes-Associated Protein is up-regulated by mechanical overload and is sufficient to induce skeletal muscle hypertrophy.

Mechanically-induced skeletal muscle growth is regulated by mammalian/mechanistic target of rapamycin complex 1 (mTORC1). Yes-Associated Protein (YAP) is a mechanically-sensitive, and growth-related, transcriptional co-activator that can regulate mTORC1. Here we show that, in skeletal muscle, mechanical overload promotes an increase in YAP expression; however, the time course of YAP expression is markedly different from that of mTORC1 activation. We also show that the overexpression of YAP induces hypertrophy via an mTORC1-independent mechanism. Finally, we provide preliminary evidence of possible mediators of YAP-induced hypertrophy (e.g. increased MyoD and c-Myc expression, and decreased Smad2/3 activity and muscle ring finger 1 (MuRF1) expression).

Smad3 induces atrogin-1, inhibits mTOR and protein synthesis, and promotes muscle atrophy in vivo.

Myostatin, a member of the TGF superfamily, is sufficient to induce skeletal muscle atrophy. Myostatin-induced atrophy is associated with increases in E3-ligase atrogin-1 expression and protein degradation and decreases in Akt/mechanistic target of rapamycin (mTOR) signaling and protein synthesis. Myostatin signaling activates the transcription factor Smad3 (Small Mothers Against Decapentaplegic), which has been shown to be necessary for myostatin-induced atrogin-1 expression and atrophy; however, it is not known whether Smad3 is sufficient to induce these events or whether Smad3 simply plays a permissive role. Thus, the aim of this study was to address these questions with an in vivo model. To accomplish this goal, in vivo transfection of plasmid DNA was used to create transient transgenic mouse skeletal muscles, and our results show for the first time that Smad3 expression is sufficient to stimulate atrogin-1 promoter activity, inhibit Akt/mTOR signaling and protein synthesis, and induce muscle fiber atrophy. Moreover, we propose that Akt/mTOR signaling is inhibited by a Smad3-induced decrease in microRNA-29 (miR-29) expression and a subsequent increase in the translation of phosphatase and tensin homolog (PTEN) mRNA. Smad3 is also sufficient to inhibit peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) promoter activity and to increase FoxO (Forkhead Box Protein, Subclass O)-mediated signaling and the promoter activity of plasminogen activator inhibitor 1 (PAI-1). Combined, this study provides the first evidence that Smad3 is sufficient to regulate many of the events associated with myostatin-induced atrophy and therefore suggests that Smad3 signaling may be a viable target for therapies aimed at preventing myostatin-induced muscle atrophy.